PROJECT SUMMARY/ABSTRACT Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) causes serious hospital-acquired infections with mortality rates exceeding 50%, and new strategies are required to treat it. The monoclonal antibody (mAb) 17H12 developed by the Fries Laboratory binds to the capsular polysaccharide (CPS) to a subset of the most prominent CR-Kp clone in the United States, ST258. The mAb has been shown to broadly react with ST258 strains of multiple serotypes, kill CR-Kp through phagocytosis and other mechanisms in vitro, and protect mice from CR- Kp infection in both an intratracheal and extra-intestinal dissemination model. However, it remains unknown how protection by this mAb occurs in vivo, and such questions are crucial if 17H12 is to be used to treat immunocompromised patients that CR-Kp primarily affects. Furthermore, our collaborations have also uncovered a synthetic oligosaccharide epitope that 17H12 recognizes and can elicit an immunogenic response to the CPS of this ST258 subset as well. This data suggests that the target of 17H12 may serve as a cheaper, more easily producible alternative to full-sized CPS that can also cover a wider range of strains, but this possibility remains to be tested. This proposal seeks to determine the means by which 17H12 mitigates CR-Kp infection in vivo, and test synthetic epitopes of 17H12 that can act as potential vaccines. I will test my central hypothesis that anti-capsular antibodies protect mice from CR-Kp extra-intestinal dissemination through the action of professional phagocytes, and that epitopes that these mAbs recognize can be used to protect against dissemination. In Aim 1, I will examine whether phagocyte populations and the Fc receptor are required for antibody-mediated protection relative to complement-mediated mechanisms. This Aim will utilize a variety of investigative tools, from flow cytometry and transformants to examine which resident phagocyte populations take up GFP-labeled CR-Kp in the presence of antibody, to the use of IgG subclass switch variants and Fc receptor knockouts in ex-vivo phagocytosis assays and in vivo infection studies. I will also apply intra-vital microscopy to observe mAb- mediated opsonophagocytosis in real time. In Aim 2 I will assess whether vaccination with small molecules that 17H12 recognizes can protect against infection by ST258 clade 2 CR-Kp. I will determine whether our synthetic oligosaccharide that binds 17H12 elicits a superior immune response to full-sized CPS through vaccination and challenge. Additionally, I will utilize phage display to identify a peptide mimic of the CPS epitope and test whether 17H12 recognizes this mimotope in the same way as it recognizes the oligosaccharide. The findings of this proposal will not only allow us to examine the role of anti-capsular immunity in the clearance of CR-Kp, but also allow us to identify patients who may benefit best from mAb therapy, promote vaccination efforts, and better understand the capsule of the ST258 clone.